Optimized Therapeutic 177Lu-Labeled PSMA-Targeted Ligands with Improved Pharmacokinetic Characteristics for Prostate Cancer

Clinical trials have shown the significant efficacy of [177Lu]Lu-PSMA-617 for treating prostate cancer. However, the pharmacokinetic characteristics and therapeutic performance of [177Lu]Lu-PSMA-617 still need further improvement to meet clinical expectations. The aim of this study was to evaluate the feasibility and therapeutic potential of three novel 177Lu-labeled ligands for the treatment of prostate cancer. The novel ligands were efficiently synthesized and radiolabeled with non-carrier added 177Lu; the radiochemical purity of the final products was determined by Radio-HPLC. The specific cell-binding affinity to PSMA was evaluated in vitro using prostate cancer cell lines 22Rv1and PC-3. Blood pharmacokinetic analysis, biodistribution experiments, small animal SPCET imaging and treatment experiments were performed on normal and tumor-bearing mice. Among all the novel ligands developed in this study, [177Lu]Lu-PSMA-Q showed the highest uptake in 22Rv1 cells, while there was almost no uptake in PC-3 cells. As the SPECT imaging tracer, [177Lu]Lu-PSMA-Q is highly specific in delineating PSMA-positive tumors, with a shorter clearance half-life and higher tumor-to-background ratio than [177Lu]Lu-PSMA-617. Biodistribution studies verified the SPECT imaging results. Furthermore, [177Lu]Lu-PSMA-Q serves well as an effective therapeutic ligand to suppress tumor growth and improve the survival rate of tumor-bearing mice. All the results strongly demonstrate that [177Lu]Lu-PSMA-Q is a PSMA-specific ligand with significant anti-tumor effect in preclinical models, and further clinical evaluation is worth conducting.


Introduction
Prostate cancer represents a significant public health problem and a heavy medical burden throughout the world. The incidence and cancer-related death of prostate cancer ranked first and second among male malignant tumors, respectively [1][2][3][4]. The options of therapeutic regimens are mainly based on the different clinical stages of patients [5,6]. Radical prostatectomy (RP) and radical radiotherapy are effective for early and localized PCa patients [7]. For patients with advanced or metastatic PCa, endocrine therapy, radiotherapy, chemotherapy and biotherapy are often applied, but the effectiveness of these available protocols is far from satisfactory; almost all patients will have disease progression in a short time [8][9][10][11][12]. Radionuclide therapy, as a novel therapeutic option that uses the biological effects of radiation emitted by radionuclides in the process of decay (α or β-) to inhibit or destroy pathological tissues, has attracted much attention in recent years. The commonly used radionuclides mainly include 131 I, 125 I, 89 Sr, 90 Y, 177 Lu, 223 Ra, etc. [13][14][15][16]. The radionuclides or the targeted compounds radiolabeled with these radionuclides can accumulate at the tumor lesion selectively, making the tumor subject to high-dose irradiation  Figure S1), respectively. Each of the radiotracers was prepared with a radiochemical purity (RCP) of more than 95% as analyzed by radio-HPLC, with retention times of 6.79 min ([ 177 Lu]Lu-PSMA-BP 29 ), 3.81 min ([ 177 Lu]Lu-PSMA-4PY) and 5.11 min ([ 177 Lu]Lu-PSMA-Q), respectively (shown in Figure 1). The molar activities (Am) were roughly calculated as 14.56 ± 3.33, 16.21 ± 5.11 and 18.42 ± 3.98 GBq/µmol.

In Vitro Cellular Studies
As human prostate cancer epithelial cell lines, 22Rv1 (mild PSMA+) and P (PSMA−) were used to evaluate the specific cell-binding affinity of the radiotracers shown in Figure 3, the uptake in 22Rv1 cells increased within the time tested, and al novel tracers revealed a substantial high uptake in 22Rv1 cells; among th [ 177 Lu]Lu-PSMA-Q showed the highest uptake in 22Rv1 cells (3.65 ± 0.27 IA%/10 6 at 2 followed by [ 177 Lu]Lu-PSMA-BP (3.18 ± 0.30 IA%/10 6 ) and [ 177 Lu]Lu-PSMA-4PY (2. 0.47 IA%/10 6 ). Uptake in 22Rv1 cells was significantly blocked by co-incubation w 2-PMPA. However, uptake of each novel tracer in PC-3 cells (lower than 0.5 IA%/10 6 a h) was much lower than that in 22Rv1 cells (p < 0.01), and could not be blocked co-incubation with 2-PMPA.

In Vitro Cellular Studies
As human prostate cancer epithelial cell lines, 22Rv1 (mild PSMA+) and PC-3 (PSMA−) were used to evaluate the specific cell-binding affinity of the radiotracers. As shown in Figure 3, the uptake in 22Rv1 cells increased within the time tested, and all the novel tracers revealed a substantial high uptake in 22Rv1 cells; among these, [ 177 Lu]Lu-PSMA-Q showed the highest uptake in 22Rv1 cells (3.65 ± 0.27 IA%/10 6 at 24 h), followed by [ 177 Lu]Lu-PSMA-BP (3.18 ± 0.30 IA%/10 6 ) and [ 177 Lu]Lu-PSMA-4PY (2.96 ± 0.47 IA%/10 6 ). Uptake in 22Rv1 cells was significantly blocked by co-incubation with 2-PMPA. However, uptake of each novel tracer in PC-3 cells (lower than 0.5 IA%/10 6 at 24 h) was much lower than that in 22Rv1 cells (p < 0.01), and could not be blocked by co-incubation with 2-PMPA.

In Vitro Cellular Studies
As human prostate cancer epithelial cell lines, 22Rv1 (mild PSMA+) and PC-3 (PSMA−) were used to evaluate the specific cell-binding affinity of the radiotracers. As shown in Figure 3, the uptake in 22Rv1 cells increased within the time tested, and all the novel tracers revealed a substantial high uptake in 22Rv1 cells; among these, [ 177 Lu]Lu-PSMA-Q showed the highest uptake in 22Rv1 cells (3.

Pharmacokinetics and Biodistribution Studies
Pharmacokinetics and biodistribution studies showed that all novel ligands could be rapidly eliminated from the blood, with clearance half-lives of 29.66 [29]

Pharmacokinetics and Biodistribution Studies
Pharmacokinetics and biodistribution studies showed that all novel ligands could be rapidly eliminated from the blood, with clearance half-lives of 29.66 [29]

Pharmacokinetics and Biodistribution Studies
Pharmacokinetics and biodistribution studies showed that all novel ligands could be rapidly eliminated from the blood, with clearance half-lives of 29.66 [29] (    As shown in Figure 5 and Table 1         Due to the high uptake in the tumor and the fast clearance rate, the tumor-to-blood (T/B), tumor-to-muscle (T/M) and tumor-to-kidney ratios were 17.59, 48. 38

Therapy Study
To evaluate antitumor efficacy, a pilot experiment was conducted using 5 groups of

Therapy Study
To evaluate antitumor efficacy, a pilot experiment was conducted using 5 groups of

Therapy Study
To evaluate antitumor efficacy, a pilot experiment was conducted using 5 groups of  The injection day was defined as Day 0, and day the mice die or reach any endpoint criterion was defined as the termination day. Tumor-bearing mice without any of the above conditions were continuously observed until Day 32.
The results showed that the weight of the mice in each experimental group decreased slightly in the short term after the administration of ligands and then remained relatively The tumor growth in the BP group was also slower than in the control group, but there was no significant difference throughout the whole experimental period (p > 0.05) (shown in Figure 8).
The injection day was defined as Day 0, and day the mice die or reach any endpoint criterion was defined as the termination day. Tumor-bearing mice without any of the above conditions were continuously observed until Day 32.
The results showed that the weight of the mice in each experimental group decreased slightly in the short term after the administration of ligands and then remained relatively stable. At Day 32, the weight of mice in the [ 177 Lu]Lu-PSMA-BP and [ 177 Lu]Lu-PSMA-4PY groups decreased compared with before treatment (p < 0.05), while there were no statistical differences in the weight of [ 177 Lu]Lu-PSMA-Q and [ 177 Lu]Lu-PSMA-617 groups before and after treatment (p > 0.05). The weight of the control group decreased significantly within the experimental period (p < 0.05).
Eight days post injection, the tumor volume of the [ 177 Lu]Lu-PSMA-Q and [ 177 Lu]Lu-PSMA-617 groups was already significantly lower than that of the control group (p < 0.05). The ratio of tumor volume at Day 8 to tumor volume at Day 0 (vt/v0) was 3.53 ± 1.14, 1.49 ± 0. 33  The tumor growth in the BP group was also slower than in the control group, but there was no significant difference throughout the whole experimental period (p > 0.05) (shown in Figure 8).

Radiotoxicity
The mice in the experimental groups were injected with 74 MBq of [ 177 Lu]Lu-PSMA-R or [ 177 Lu]Lu-PSMA-617, and there was no death within the observation period. There were no significant differences in diet, excretion, activity, mental state, skin condition, and weight change between the experimental group and the control group (p > 0.05). No significant differences were observed in the size, color, shape, and texture of the organs among the five groups. , respectively. There were no significant differences in the results of the HE staining of the sections of the main organs among the five groups; moreover, no obvious toxic damage, such as necrosis and fibrosis, was observed under the microscope. The blood routine indexes of mice in each group remained within the normal reference value range on the 14th and 28th days after the injection of radioactive drugs (shown in Figure  10).

Radiotoxicity
The mice in the experimental groups were injected with 74 MBq of [ 177 Lu]Lu-PSMA-R or [ 177 Lu]Lu-PSMA-617, and there was no death within the observation period. There were no significant differences in diet, excretion, activity, mental state, skin condition, and weight change between the experimental group and the control group (p > 0.05). No significant differences were observed in the size, color, shape, and texture of the organs among the five groups. The kidney weights of the mice in each group were 0.191 ± 0. , respectively. There were no significant differences in the results of the HE staining of the sections of the main organs among the five groups; moreover, no obvious toxic damage, such as necrosis and fibrosis, was observed under the microscope. The blood routine indexes of mice in each group remained within the normal reference value range on the 14th and 28th days after the injection of radioactive drugs (shown in Figure 10).

Discussion
Prostate cancer is one of the most highly prevalent malignancies in men. By 2020, prostate cancer had become the most commonly diagnosed cancer among men in 112 countries [30]. Since the development of advanced prostate cancer is mainly driven by the androgen signaling pathway, hormone therapy is the most commonly used treatment for patients with prostate cancer who have lost the opportunity for radical surgery and radical radiotherapy. In the early stage of anti-androgen therapy, prostate cancer is most effectively controlled; however, with extension of the treatment period, almost all patients experience progression that is difficult to suppress with hormone therapy [8,9]. During the course of anti-androgen therapy, testosterone remains at depleted levels, and prostate cancer progression within this period is referred to as castration-resistant prostate cancer (CPRC) [31]. Currently, docetaxel, a paclitaxel antineoplastic drug, is mostly used as a first-line treatment option for CPRC. However, after multicycle chemotherapy, most patients experience significant toxic side effects and develop drug resistance, and the growth and invasion rate of tumors increases significantly again [32][33][34]. The new generation of antiandrogen drugs, abiraterone and enzalutamide, have improved progression-free and overall survival in patients with CRPC to some extent, but the improvement of the overall prognosis was limited. In addition, studies found that resistance to either abiraterone or enzalutamide was always accompanied by resistance to another new generation anti-androgen drug and docetaxel [35][36][37][38]. On the basis of the above points, the treatment of CRPC remains an important and difficult problem to be solved.
Prostate specific membrane antigen (PSMA), which is specifically overexpressed on almost all prostate cancer cell membranes, is considered to be an ideal target for the diagnosis and treatment of prostate cancer; PSMA-mediated radionuclide therapy (PRRT) has also been a research hotspot in recent years. A combination of β-rays-emitted radionuclides and PSMA-targeted ligands can bind to the surface of PCa cells and further internalize into cells, thus exerting the effect of killing the tumor. 177 Lu is the most commonly used radionuclide for radiotherapy; it emits β-rays of moderate energy (Emax = 0.5 MeV), with a tissue penetration range of approximately 2 mm and a physical half-life of 6.7 days, while the γ-photons emitted by 177 Lu can also be used for SPECT imaging to monitor the curative effect before and after treatment. Studies have confirmed the safety and effectiveness of 177 Lu-labeled PSMA ligands such as [ 177 Lu]Lu-PSMA-617 and [ 177 Lu]Lu-PSMA-I & T in the treatment of prostate cancer, but their pharmacokinetic properties for PCa treatment, among others, still need to be improved [39,40]. In this study, we evaluated and compared the therapeutic potential of the PSMA ligands The stronger hydrophilicity facilitated the clearance of the ligands from blood, normal tissues, and organs, reducing the possibility of toxic reactions. Subsequent animal toxicity experiments also proved their safety again. After euthanasia and autopsy of the mice, there was no significant difference in the size, color, texture, and weight of the main organs of the mice in either the experiment or control group. The results of organ HE staining showed that there were no abnormalities in organs among five groups, especially in the kidney and salivary glands, which were vulnerable to radiation. No abnormality was found in the blood routine tests in each group.
The purpose of radionuclide endoradiotherapy is to maximize the absorbed dose of radionuclide by tumors, while ensuring that uptake of other normal tissues is within a reasonable range. In the in vitro cellular uptake assay, all novel ligands were specifically accumulated in 22Rv1 cells with PSMA-positive expression, among which [ 177 Lu]Lu-PSMA-Q showed the highest uptake, followed by [ 177 Lu]Lu-PSMA-BP and [ 177 Lu]Lu-PSMA-4PY; there was almost no uptake in PC-3 cells. Further biodistribution and SPECT imaging results showed that the three novel ligands could be specifically uptaken by 22Rv1 tumors. The uptake in the tumors was higher than in other normal organs. The salivary glands, which were vulnerable to radiation damage, showed low uptake of all ligands, with uptake values less than 0.5 %ID/g 24 h p.i. in the biodistribution experiment. The tumor, kidney, and bladder were clearly visualized on SPECT images at 24 h p.i., and other background tissues showed extremely low uptake. The results showed that the body weight of mice in the treatment and control groups decreased to a certain extent in a short period. This was due to the tumor itself being in a multiplicative stage of growth; cell growth was vigorous and a lot of nutrients were needed. However, the weight of tumorbearing mice in the Q group gradually stabilized around 8 d and increased slightly around 20 d after therapy. The weight of mice in the 617 group decreased slightly and began to stabilize around 14 d p.i. The weight of the BP and 4PY groups continuously decreased; however, the decrease was smaller than that of the control group, indicating that tumor cell growth activity in the treatment groups had been inhibited to some extent. However, mice in the control group continued to lose weight, and symptoms similar to cachexia, such as poor mental status, reduced mobility, and reduced food intake, appear in later stages. At the end of the treatment experiment, the weight of the mice in every treatment group was higher than that of the control group. In terms of tumor growth, compared with the control group, the tumor growth rate of all treatment groups was significantly slower. ]Lu-PSMA-617 at the termination were 80%, 20%, 50% and 60%. The survival rate of each group in the experimental group was significantly higher than that of the control group. Within the four treatment groups, survival rates were not statistically different among Q, 617 and 4PY groups (p > 0.05); however, the Q and 617 groups were higher than the BP group (p < 0.05), and there was no difference between the 4PY and BP groups (p > 0.05).
In terms of the effect of structural modifications on therapeutic efficacy, incorporation of the 4-pyridyl, 3-quinoline, or biphenyl moiety enhances the hydrophilicity of the ligand, especially the 4-pyridyl. With the guarantee of the affinity of the ligands, a higher hydrophilicity indicates a faster blood clearance rate and a higher tumor-to-background ratio, which may be beneficial to tumor treatment efficacy and reduce toxic side effects in accordance with our expectation. However, the relatively low binding affinity and excessive hydrophilicity of [ 177 Lu]Lu-PSMA-4PY lead to low uptake in tumors and rapid clearance of [ 177 Lu]Lu-PSMA-4PY, not only from blood and normal organs, but also from the tumor, resulting in undesirable therapeutic efficacy. Compared with 4-pyridine, the incorporation of 3-quinoline affects the affinity of the ligand to a small extent, which is coupled with its moderate hydrophilicity, resulting in an ideal outcome. In addition to the above, actually, because of the complexity of the in vivo environment, the results of in vivo therapeutic studies are difficult to predict purely based on the characteristics of ligands and the results of in vitro studies. Regardless of the modification of the structure, the results of in vivo therapeutic studies are the ultimate reflection of whether the modification meets initial expectations.
However, there are still limitations in this study. First, the inhibitory effect of ligands on tumors in this study is lower than that reported in some of the literature. The reasons for this are, on the one hand, the large size of the tumor used for the evaluation of the efficacy in this study compared with that reported in the literature (100 mm 3 vs. 20 mm 3 ), and the fact that larger tumors are somewhat weakened by the therapeutic effect of radionuclides due to possible necrosis in their centers and the difficulty of penetration of radiation; on the other hand, the 22Rv1 cells have a lower level of PSMA expression than LNCaP and PC-3 PIP cells. Second, in the evaluation of drug toxicity, the observation period was relatively short; only the absence of acute toxic effects of the drugs could be confirmed. Third, multiple cycles of treatment are commonly used in clinical patients; however, in this study, only a single dose was administered, so the safety and efficacy of multiple treatments still need further study. Fourth, the absorbed dose of the ligands in various organs should be estimated to ensure that the dose in normal tissues and organs is within an acceptable range. However, as a preclinical study, the basic experimental results provided by this study confirmed the potential of [ 177 Lu]Lu-PSMA-Q for prostate cancer treatment; this was sufficient to support clinical transformation and large-scale prospective studies to further validate its effectiveness, and to ensure that the effectiveness is not inferior to that of [ 177 Lu]Lu-PSMA-617, while producing less radiation damage and toxic effects.

Materials and Methods
All chemicals, reagents, and solvents for the synthesis and analysis were of analytical grade (all purchased from Maclin Biochemical Technology Co., Ltd. (Shanghai, China)). 177 LuCl 3 solution was purchased from ITG Isotope Technologies Garching GmbH (München, Germany). All animal studies were performed according to a protocol approved by the Animal Care and Use Committee of Chinese PLA General Hospital.

Partition Coefficient
The octanol-water partition coefficient was determined by adding [ 177 Lu]Lu-PSMA-R (0.37 MBq in 100 µL), 2.9 mL of phosphate-buffered saline (0.1 M, pH 7.4) and 3 mL of octanol to a tube. The mixture was vortexed for 2 min and centrifuged (3000 rpm × 5 min). Then, 3 samples (1 mL) from each phase were collected and the radioactivity was measured by a γ-counter (Wallac 2480 Wizard, MA), respectively. The experiment was carried out in triplicate and repeated 3 times. Log p value was calculated using log p = log (CPM for octanol/CPM for water) and reported as Log p ± SD.

Cell Lines and Culture Condition
Cell lines were kindly provided by the stem cell bank at the Chinese Academy of Sciences. The human prostate cancer cell lines 22Rv1 (mild PSMA+) and PC3 (PSMA-) were cultured in RPMI 1640 medium (Gibco, New York, NY, USA) and supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) in a humidified incubator at 37 • C under 5% CO 2 . Each cell line was passaged to around 80-90% confluence, and harvested using trypsin-ethylenediaminetetraacetic acid (trypsin-ETDA; 0.25% trypsin, 0.02% ETDA, all from Gibco).

Cell Binding and Internalization Studies
Cells were seeded in 24-well plates (1 × 10 5 cells in 1 mL medium/well) and incubated at 37 • C/5% CO 2 overnight; the medium was changed 2 h before the experiment. Each well was then added with 177 Lu-PSMA-R (0.074 MBq in 100 µL saline) and incubated at 37 • C/5% CO 2 for 24 h. After incubation, the medium was removed and the cells were washed twice with ice-cold phosphate-buffered saline (0.5 mL) at each time point; finally, the cells were lysed by NaOH (0.5 M, 0.5 mL). 2-(phosphonomethyl) pentane-1,5dioic acid (2-PMPA) solution (2 µg/well) was used for the blocking experiment. For the internalization experiment, cells were incubated with glycine-HCL in PBS (50 nM, PH 2.8, 0.5 mL) for 5 min at room temperature, then washed twice with ice-cold PBS (0.5 mL) and lysed by NaOH (0.5 M, 0.5 mL). The radioactivity of cells was measured using a γ-counter. The studies were performed in triplicate and the result was reported as percentage injected activity (%IA)/10 6 cells.

Biodistribution
The 22Rv1 tumor-bearing mice were injected with [ 177 Lu]Lu-PSMA-R or [ 177 Lu]Lu-PSMA-617 (4 groups, n = 3/group, 0.74 MBq in 150 µL per mouse). Mice were sacrificed at 24 h p.i., tumors and interested organs were harvested, weighed and measured by a γ-counter, and the results were expressed as the percentage of injected dose per gram (ID%/g).

Radiotoxicity
Healthy ICR male mice were injected with [ 177 Lu]Lu-PSMA-R or [ 177 Lu]Lu-PSMA-617 (74 MBq in 150 µL) or saline (150 µL) for the radiotoxicity evaluation (5 groups, n = 15/group). At 14 d post injection, venous blood was drawn from the tail vein for the routine blood test; subsequently, 5 of the mice in each group were euthanized and the main organs were harvested and fixed in 4% formalin in PBS. Organ samples were trimmed and embedded in paraffin. Then, histological sections were prepared, stained with hematoxylin-eosin and assessed under a light microscope. At 28 d post injection, the routine blood test was carried out again in the remaining mice in each group.

Statistical Analysis
All quantitative data were expressed as mean ± SD. The normality of the data was assessed by the Shapiro-Wilk test, followed by the two-tailed Student's t-test or the Mann-Whitney U test. A p value of <0.05 was considered statistically significant. Statistical analyses were performed using SPSS software 22.0 (IBM Corp., Armonk, NY, USA) and Prism 8 software (GraphPad Software, San Diego, CA, USA).

Conclusions
In this study, we evaluated the radiotherapy efficacy of three novel 177 Lu-labeled ligands selected from our previous study. [ 177 Lu]Lu-PSMA-Q exhibited the highest affinity and excellent pharmacokinetic characteristics, resulting in a satisfactory tumor inhibition effect which was not inferior to [ 177 Lu]Lu-PSMA-617 and is worth further study.

Conflicts of Interest:
The authors declare no competing financial interest.